1. Field of the Invention
The present invention relates to a zearalenone-inducible promoter originated from Gibberella zeae, recombinant vectors comprising the promoters, methods of producing proteins and detecting plants contaminated with zearalenone.
2. Description of the Related Art
The ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is an important plant pathogen that causes fusarium ear rot disease on maize and head blight on barley, wheat, and rice (17). This fungus produces mycotoxins, such as trichothecenes and zearalenone (ZEA), which are harmful to humans and animals. Trichothecenes are sesquiterpenoids that are potent inhibitors of eukaryotic protein biosynthesis and cause food refusal, diarrhea, emesis, alimentary hemorrhaging, and contact dermatitis in animals (3). In addition, trichothecenes are virulence factors in plants (26).
ZEA [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcyclic acid lactone] is a polyketide metabolite (3) that causes estrogenic disorders in laboratory rats, mice, and farm-raised swine that have ingested fusarium-contaminated maize, wheat, and barley (22). ZEA biosynthesis genes in G. zeae are located in a gene cluster that contains two polyketide synthase genes, one putative transcription factor, and one putative isoamyl alcohol oxidase gene (4, 12, 19). ZEA-nonproducing mutants generated from G. zeae field strains do not have any noticeable phenotypic changes except for a loss of ZEA production (4, 12, 19). The biological functions of ZEA in G. zeae have not been characterized to date.
Molecular studies of G. zeae have been widely performed by many research groups since the genome was sequenced by the Broad Institute of Cambridge, Mass. Molecular manipulations, including targeted gene deletion, gene overexpression, and gene fusion to green fluorescent protein (GFP), are relatively easy to perform with this fungus. In addition, mutant collections of G. zeae generated through restriction enzyme-mediated integration mutagenesis are currently available (6, 29), and the functions of genes related to toxin biosynthesis, sexual reproduction, pigmentation, and virulence in the fungus have been well characterized (3, 9, 11, 14, 37). However, a conditional expression system that uses a conditional promoter for the study of essential genes and induction of transgenes is currently not available in G. zeae. 
Regulation of gene expression by conditional promoters, including copper-, cadmium-, thiamine-, and alcohol-responsive promoters, has been developed in various organisms (23, 25, 30, 34-35). These systems have been used as tools for studying the molecular regulation of target gene expression and protein production of genes in industrial fungi (20, 39). Further, recombinant bacterial and fungal strains that possess a metal-inducible promoter have been used as biosensors for monitoring heavy metal contamination (25, 33). A woundinducible promoter was also recently used to generate transgenic plants that can recognize fungal infections (36).
Previously, the present inventors performed a microarray analysis to characterize the biological functions of ZEA in G. zeae and found that the transcripts of certain genes, including ZEA biosynthetic genes, were highly elevated after ZEA treatment. The present inventors hypothesized that the promoters of genes upregulated by ZEA treatment could be used as inducible promoters for the conditional expression of target genes in response to ZEA treatment. From our analysis of genes upregulated by exogenous treatment of ZEA, one ZEA response gene (Broad Institute; FGSG—04581.3) was selected for further study based on an expression level that was 50 times higher than that of the control in cultures containing ZEA. The gene was designated a ZEA response gene (ZEAR). The present inventors have successfully identified a ZEA-inducible promoter and applied it as a tool for regulating gene expression in G. zeae. 